Methods and systems for screening therapeutic compositions for treating age-related macular degeneration

ABSTRACT

Methods and systems for screening therapeutic compositions for the treatment of age-related macular degeneration (AMD) or for the prevention of progression of AMD, wherein retinal pigment epithelium cells (RPE) are cultured and the level of hypoxia-induced exosome release from the RPE cells is subsequently measured. In the present invention, the level of hypoxia-induced exosome release can be correlated with AMD onset and severity.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a non-provisional and claims benefit of U.S.Provisional Application No. 63/037,886 filed Jun. 11, 2020, thespecification(s) of which is/are incorporated herein in their entiretyby reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. R01EY026544, awarded by National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is directed to therapeutic compositions fortreating, preventing progression of, or ameliorating symptoms ofage-related macular degeneration (AMD), more particularly to screeningcompositions for therapeutic potential for treating AMD.

Background Art

Age-related macular degeneration (AMD) is a loss of central vision thatoccurs in either dry, atrophic or wet, exudative (neovascular) forms.Most people with macular degeneration have the atrophic form. Accordingto data from the American Optometrist Association, AMD is the leadingcause of severe vision loss in adults over age 50. While there is nospecific treatment for atrophic AMD, studies have shown a potentialbenefit from vitamin supplements, a healthy diet, cessation of smoking,and perhaps intraocular injections. Wet AMD is frequently treated withintraocular injections to inhibit vascular endothelial growth factor(VEGF) activity. However, this line of treatment is expensive.

BRIEF SUMMARY OF THE INVENTION

It is an objective of the present invention to provide methods thatallow for the identification of therapeutic compositions for thetreatment of AMD, as specified in the independent claims. Embodiments ofthe invention are given in the dependent claims. Embodiments of thepresent invention can be freely combined with each other if they are notmutually exclusive.

In some aspects, the present invention features methods and systems forscreening agents or therapeutic compositions (e.g., drugs) for thetreatment and/or prevention of age-related macular degeneration (AMD),e.g. prevention of development of AMD, prevention of progression of AMD.

Briefly, retinal pigment epithelium cells (RPE) may be cultured and thelevel of exosome release from the RPE cells may be measured afterincubation in a hypoxic environment with candidate therapeuticcompositions. The level of exosome release can be correlated with AMDonset and severity. Thus, if a therapeutic composition can reduceexosome release in these RPE assays, then it may be a good candidatedrug for treating AMD, preventing its development or progression, orameliorating symptoms. While exosomes range from 40-140 nm in diameter,these RPE assays are designed to evaluate the cargo and concentration ofall extracellular vesicles.

Inventors surprisingly discovered that massive exosome release inretinal pigment epithelial cells (RPEs) is caused by hypoxia and isrelated to the development and progression of AMD. The current art hasno teachings of using hypoxia to release exosomes from RPE cells.Furthermore, the cargo carried by exosomes may identify the form of AMDin a patient. For example, exosomes from patients with dry AMD containnon-angiogenic biomarkers and can be characterized by reduced or absentpigment epithelial derived factor (PEDF), while exosomes from patientswith wet AMD contain more angiogenic and pro-inflammatory biomarkerssuch as vascular endothelial growth factor (VEGF) or IL-6. Further,agents that block or reduce RPE exosome release (e.g., hypoxia-inducedRPE exosome release) are believed to be candidates for treating AMDand/or preventing the development or progression of AMD.

The present invention features a rapid method for screening therapeuticcompounds that may be used for treating AMD. Current methods foridentifying compounds to treat AMD may take up to several years. Thereis no treatment for dry AMD because it progresses slowly, thus anydouble blind clinical trial for treating dry AMD needs to be at least7-9 years to see a minor effect. Neovascular AMD is faster to see aneffect largely because the disease process is faster. Once a patientconverts from dry AMD to wet AMD, they need to be treated or they willlose significant vision in a year. The methods described herein may take30 minutes to 4 hours to screen compounds that may be used to treat AMD.In some aspects, for example, the method may use a screen utilizingexosome release from cultured RPE cells in response to hypoxia, with thehypoxic episode lasting just 30 minutes.

The present invention also describes regulating the G-protein coupledreceptor (GPCR) GPR143, which is believed to be a target for regulatingexosome release and the development and/or progression of AMD. Forexample, the present invention describes treating AMD and/or preventingthe development or progression of AMD by introducing GPR143 agonistssuch as but not limited to L-DOPA.

The methods herein to screen and identify drug candidates for AMD arescalable to industry levels. For example, the methods here may be usedto screen hundreds or thousands of compounds. Candidates from the screencan be further tested, for example tested using hypoxia and laserinduced neovascularization animal models as they are moved towards theclinic, etc.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. Additional advantages and aspects ofthe present invention are apparent in the following detailed descriptionand claims.

Terms

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which a disclosed invention belongs. The singularterms “a,” “an,” and “the” include plural referents unless contextclearly indicates otherwise. Similarly, the word “or” is intended toinclude “and” unless the context clearly indicates otherwise. The term“comprising” means that other elements can also be present in additionto the defined elements presented. The use of “comprising” indicatesinclusion rather than limitation. Stated another way, the term“comprising” means “including principally, but not necessary solely”.Furthermore, variation of the word “comprising”, such as “comprise” and“comprises”, have correspondingly the same meanings. In one respect, thetechnology described herein related to the herein describedcompositions, methods, and respective component(s) thereof, as essentialto the invention, yet open to the inclusion of unspecified elements,essential or not (“comprising”).

All embodiments disclosed herein can be combined with other embodimentsunless the context clearly dictates otherwise.

Suitable methods and materials for the practice and/or testing ofembodiments of the disclosure are described below. Such methods andmaterials are illustrative only and are not intended to be limiting.Other methods and materials similar or equivalent to those describedherein can be used. For example, conventional methods well known in theart to which the disclosure pertains are described in various generaland more specific references, including, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, 1989; Sambrook et al., Molecular Cloning: A LaboratoryManual. 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates, 1992 (andSupplements to 2000); Ausubel et al., Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlowand Lane, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, 1999, Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray,The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog(Ambion, Austin, Tex.), the disclosures of which are incorporated intheir entirety herein by reference.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety for allpurposes. In case of conflict, the present specification, includingexplanations of terms, will control.

Although methods and materials similar or equivalent to those describedherein can be used to practice or test the disclosed technology,suitable methods and materials are described below. The materials,methods, and examples are illustrative only and not intended to belimiting.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

As used herein, the term “disease” or “disorder” or “condition” refersto any alteration in state of the body or of some of the organs,interrupting or disturbing the performance of their functions and/orcausing symptoms such as discomfort, dysfunction, distress, or evendeath to the person afflicted or those in contact with a person. Adisease or disorder or condition can also relate to a distemper, ailing,ailment, malady, disorder, sickness, illness, complaint, indispositionor affliction.

As used herein, the terms “treat” or “treatment” or “treating” refers toboth therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow the development of the disease,such as slow down the development of a disorder, or reducing at leastone adverse effect or symptom of a condition, disease or disorder, e.g.,any disorder characterized by insufficient or undesired organ or tissuefunction. Treatment is generally “effective” if one or more symptoms orclinical markers are reduced as that term is defined herein.Alternatively, a treatment is “effective” if the progression of adisease is reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or decrease of markers of the disease, but alsoa cessation or slowing of progress or worsening of a symptom that wouldbe expected in absence of treatment. Beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (e.g., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment.

A “patient” is a subject afflicted with a disease or disorder. The term“patient” includes human and veterinary subjects.

The terms “administering” and “administration” refer to methods ofproviding a pharmaceutical composition to a subject. Such methods arewell known to those skilled in the art and include, but are not limitedto, administering the compositions suitable for oral (including buccaland sublingual), nasal, ocular (including subconjunctival, intravitreal,retrobulbar, intracameral), or parenteral (including intra-arterial,subcutaneous and intravenous), or the like.

As described above, the compositions can be administered to a subject ina pharmaceutically acceptable carrier. By “pharmaceutically acceptable”is meant a material that is not biologically or otherwise undesirable,i.e., the material may be administered to a subject without causing anyundesirable biological effects or interacting in a deleterious mannerwith any of the other components of the pharmaceutical composition inwhich it is contained. The carrier would naturally be selected tominimize any degradation of the active ingredient and to minimize anyadverse side effects in the subject, as would be well known to one ofskill in the art.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. Typically, an appropriate amount of apharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of the pharmaceutically-acceptablecarrier include, but are not limited to, saline, Ringer's solution anddextrose solution. The pH of the solution is preferably from about 5 toabout 8, and more preferably from about 7 to about 7.5. Further carriersinclude sustained release preparations such as semi-permeable matricesof solid hydrophobic polymers containing the disclosed compounds, whichmatrices are in the form of shaped articles, e.g., films, liposomes,microparticles, or microcapsules. It will be apparent to those personsskilled in the art that certain carriers can be more preferabledepending upon, for instance, the route of administration andconcentration of composition being administered. Other compounds can beadministered according to standard procedures used by those skilled inthe art.

Pharmaceutical formulations can include additional carriers, as well asthickeners, diluents, buffers, preservatives, surface active agents andthe like in addition to the compounds disclosed herein. Pharmaceuticalformulations can also include one or more additional active ingredientssuch as antimicrobial agents, anti-inflammatory agents, anesthetics, andthe like.

Pharmaceutical formulations may include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, fish oils, and injectable organic-esters such as ethyl oleate.Aqueous carriers include water, alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Additionalnon-limiting examples include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, fixed oils,fluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases and the like. Otherpharmaceutical formulations include, but are not limited to, powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets, or tablets. Thickeners, flavorings, diluents,emulsifiers, dispersing aids or binders may be desirable.

As used herein, the term “artificially inducing” refers to exposing thecultured RPE cells to an artificial stressor. Non-limiting examples ofartificial stressors include a hypoxic environment or starvation.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The patent application or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

The features and advantages of the present invention will becomeapparent from a consideration of the following detailed descriptionpresented in connection with the accompanying drawings in which:

FIG. 1 shows a flow chart of the method of the present invention foridentifying a therapeutic composition for treating or preventingprogression of age-related macular degeneration (AMD).

FIG. 2 shows a schematic view of exosome isolation using differentialultracentrifugation. Exosomes can be quantitated using total exosomeprotein (e.g., silver stain for SDS-PAGE, UV absorbance, etc.), orexosome counting (e.g., nanoparticle tracking analysis), or otherappropriate means.

FIG. 3A shows RPE-exosome release in response to L-DOPA.

FIG. 3B shows a schematic view of RPE-exosome release in response toL-DOPA.

FIG. 4A shows RPE exosome release in response to dopamine. Error barsrepresent SEM.

FIG. 4B shows RPE exosome release in response to D2R stimulation. Errorbars represent SEM. D2R agonist=bromocriptine; D2Rantagonist=raclopride.

FIG. 5 shows exosome release in response to hypoxia. Exosome releaseincreases the longer RPE undergoes hypoxia. Exosomes can be quantifiedby unbiased protein staining of SDS-PAGE gels. FIG. 5 shows thecomposite analysis of 7 experiments in which RPE tissue isolates weretreated in triplicate with DMEM 15 hours apart or 24 hours apart. Meanintegrated optical density (IOD)+/−SEM, n=21. *P<0.05 **P, 0.01,unpaired t test.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to FIG. 1, the present invention features methods andsystems for identifying a therapeutic composition effective for treatinga condition caused by increased levels of exosomes released from retinalpigment epithelial (RPE) cells. In some embodiments, the conditioncaused by increased levels of exosomes released from (RPE) cells isage-related macular degeneration.

In some embodiments, the method comprises artificially inducing exosomerelease from cultured RPE cells, incubating the cultured RPE cells witha therapeutic composition, and measuring a level of exosome release fromthe RPE cells. Without wishing to limit the present invention to anytheory or mechanism, a therapeutic composition is identified as beingeffective for treating the condition if the level of exosome releasefrom RPE cells is at least about 10% lower than the predeterminedthreshold.

In one embodiment, the predetermined threshold is determined bymeasuring a level of exosome release from a control. In someembodiments, the predetermined threshold is a level of exosome releasemeasured from cultured RPE cells in which exosome release was induced,and then incubated in media without the therapeutic composition. Inpreferred embodiments, conditions for determining the predeterminedthreshold are the same as those used in testing the therapeuticcomposition. Non-limiting examples of conditions include incubation timeand the artificial stressor used to induce exosome release. In someembodiments, the therapeutic composition is identified as beingeffective if the level of exosome release from RPE cells is at leastabout 10% lower than the predetermined threshold. In some embodiments,the predetermined threshold is a laboratory standard. In otherembodiments, the predetermined threshold is an industry standard. Insome embodiments, the therapeutic composition is an activator of GPR143.

In some embodiments, exosome release is induced by an artificialstressor. Non-limiting examples of the artificial stressor include ahypoxic environment or starvation. In certain embodiments, the hypoxicoxygen environment is an environment with a concentration of oxygen ofabout 5%. In certain embodiments, the hypoxic oxygen environment is anenvironment with a concentration of oxygen of about 10%. In certainembodiments, the hypoxic oxygen environment is an environment with aconcentration of oxygen of about 15%. In certain embodiments, thehypoxic oxygen environment is an environment with a concentration ofoxygen from 5 to 10%. In certain embodiments, the hypoxic oxygenenvironment is an environment with a concentration of oxygen from 2 to15%. In some embodiments, starvation may be induced by incubating theRPE cells with low glucose media. In certain embodiments, the RPE cellsare incubated in media with 5 mM glucose to induce starvation. In otherembodiments, the RPE cells are incubated in media with 2.5 mM glucose toinduce starvation. In some embodiments, the RPE cells are incubated inmedia with 1.5 mM glucose to induce starvation. In one embodiment, theRPE cells are incubated in media with between 1.5 to 5 mM glucose toinduce starvation.

In some embodiments, the method is a high throughput screen. In otherembodiments, the RPE cells are cultured in a multi-well plate. Examplesof multi-well plates include, but are not limited to, 96 well plates or384 well plates. In one embodiment, the RPE cells are cultured in amonolayer. In some embodiments, exosome release is induced for a firsttime period ranging from about 12-24 hours. In another embodiment, thecultured RPE cells are incubated for a second time period ranging fromabout 15-60 minutes. In other embodiments, the second time period mayrange from about 30 minutes to about 4 hours. The present invention isnot limited to the aforementioned periods of time for incubating thetherapeutic composition. In some embodiments, the media may betyrosine-free, serum-free, exosome-free media (e.g., tyrosine-free,serum-free, exosome-free DMEM).

In further embodiments, the method may comprise collecting a media inwhich the RPE cells are incubated. In yet another embodiment, the methodmay further comprise isolating exosomes from the media collected.Without wishing to limit the present invention to any theory ormechanism, the exosomes may indicate which type of AMD a patient has.For example, exosomes from patients with dry AMD contain non-angiogenicbiomarkers and are expected to have reduced or absent PEDF, whereasexosomes from patients with wet AMD contain more angiogenic andpro-inflammatory biomarkers, such as VEGF and IL-6.

Exosomes may be quantitated using total exosome protein (e.g., silverstain for SDS-PAGE, UV absorbance, etc.), or exosome counting (e.g.,nanoparticle tracking analysis), or other appropriate means. In certainembodiments, measurement of the level of exosome release is performed bymeasuring an absorbance at 260/280 (A_(260/280)) of media in which theRPE cells are cultured. The present invention is not limited to anyparticular method for measuring levels of exosomes.

The present invention features a composition for treating a conditioncaused by increased levels of exosomes released from retinal pigmentepithelial (RPE) cells. In some embodiments, the composition isidentified for treating the condition by inducing exosome release fromcultured RPE cells, incubating the cultured RPE cells with a therapeuticcomposition, and measuring a level of exosome release from the RPEcells. Without wishing to limit the present invention to any theory ormechanism, a therapeutic composition is identified as being effectivefor treating the condition if the level of exosome release from RPEcells is lower than the predetermined threshold. In some embodiments,the therapeutic composition is identified as being effective fortreating the condition if the level of exosome release from RPE cells isat least about 10% lower than the predetermined threshold.

In some embodiments, the present invention features a method ofpreventing progression of dry age-related macular degeneration (AMD) towet AMD in a subject in need thereof. The method comprises administeringto the subject a therapeutically effective amount of a therapeuticcomposition identified by the methods described herein. In anotherembodiment, the present invention features a method for treating AMD ina subject in need thereof. The method comprises administering to thesubject a therapeutically effective amount of a therapeutic compositionidentified by the methods described herein. In preferred embodiments,the therapeutic composition blocks or reduces exosome release in retinalpigment epithelial (RPE) cells.

In some embodiments, the therapeutic compositions are administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. One of ordinaryskill in the art of treating such diseases is typically able, withoutundue experimentation and in reliance upon personal knowledge and thedisclosure of this application, to ascertain a therapeutically effectiveamount of the therapeutic compositions.

In some embodiments, the therapeutic compositions are administered aspharmaceutical formulations including those suitable for oral (includingbuccal and sublingual), nasal, ocular (including subconjunctival,intravitreal, retrobulbar, intracameral), or parenteral (includingintra-arterial, subcutaneous and intravenous) administration, in a formsuitable for administration by inhalation or insufflation, or the like.

In one embodiment, the therapeutic composition may be administered to apatient intranasally, e.g. by using a nasal spray, atomizer, dropper, orsyringe. In other embodiments, the therapeutic composition may beadministered to a patient orally, e.g. by pill or lozenge form. In yetanother embodiment, the therapeutic composition may be administered to apatient in an intravenous dosage. In some embodiments, the therapeuticcomposition may be administered to a patient ocularly, e.g. by eyedrops.

The present invention features methods and systems for screening agentsor therapeutic compositions (e.g., drugs) for the treatment and/orprevention of age-related macular degeneration (AMD), e.g. prevention ofdevelopment of AMD, prevention of progression of AMD.

Retinal pigment epithelium cells (RPE) may be cultured and the level ofhypoxia-induced exosome release from the RPE cells is subsequentlymeasured after incubation with candidate therapeutic compositions atdifferent oxygen concentrations (e.g., normal oxygen versus an oxygenlevel considered to provide a hypoxic environment). For example,monolayers of RPEs (e.g., pigmented RPEs) may be grown. In someembodiments, RPE are left undisturbed at confluence for a period oftime, e.g., a minimum of 2-4 months, while maintained in low tyrosinemedium. Media may include a tyrosine-free, serum-free, exosome-freemedia (e.g., tyrosine-free, serum-free, exosome-free DMEM).

A portion of cells may then be placed in a hypoxic environment (e.g., 5%02 concentration) for 24 hours to induce exosome release, and a portionmay be placed in an environment with a normal oxygen concentration. Acandidate therapeutic composition may be introduced to the cells for aperiod of time, e.g., 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90minutes, 2 hours, 3 hours, 4 hours, or a period of time between 15minutes to 4 hours, etc. The present invention is not limited to theaforementioned periods of time for incubating the candidate therapeuticcomposition. Following incubation with the candidate therapeuticcomposition, the level of exosomes may be measured. In certainembodiments, the level of protein in the media is measured directly,e.g., without purifying any particular proteins, etc. In certainembodiments, the media is collected, exosomes are isolated, and levelsof exosomes are measured. Methods of isolating exosomes are well knownto one of ordinary skill in the art (e.g., polyethylene glycolprecipitation). The present invention is not limited to theaforementioned methods of measuring levels of exosomes.

Exosomes may be quantitated using total exosome protein (e.g., silverstain for SDS-PAGE, UV absorbance, etc.), or exosome counting (e.g.,nanoparticle tracking analysis), or other appropriate means. In certainembodiments, total exosome protein is measured using direct 260/280absorbance. The present invention is not limited to any particularmethod for measuring levels of exosomes.

The exosome protein levels may be compared between the drug-treatedcells and the control cells. Further, the level of exosome release canbe correlated with AMD onset and severity. Thus, if a therapeuticcomposition can reduce exosome release in RPE assays, then it may be agood candidate drug for treating AMD, preventing its development orprogression, or ameliorating symptoms.

The methods of the present invention may be carried out using anyappropriate cell culture plates, including multi-well plates such as12-well plates, 48-well plates, 98-well plates, 384-well plates, etc.Thus, the present invention provides for a high-throughput screeningmethod.

Table 1 below illustrates one embodiment of the screening method of thepresent invention. The present invention is not limited to the methods,assays, conditions, or compositions described herein.

TABLE 1 Step Description 1 Grow monolayers of pigmented RPE 2 Placecells in hypoxic O₂ environment for a period of time to induce exosomerelease 3 Add test compound (e.g., drug) to be tested, incubate for aperiod of time 4 Collect conditioned media with exosomes 5 Isolateexosomes by any appropriate method (eg., polyethylene glycolprecipitation, exosome isolation kit, etc.) 6 Measure protein inisolated exosomes (any appropriate method to measure, e.g., direct260/280 absorbance) 7 Compare exosome protein amounts between drugtreated and control media (or compare exosome protein amounts betweendrug treated media and a predetermined threshold)

In certain embodiments, the RPE cells must be pigmented, so the cellsmay be left undisturbed at confluence for a period of time, e.g.,minimum of 2-4 months. Note that tyrosine may cause inactivation of theGPR143 receptor, so the cells may be maintained in low tyrosine mediumsupplemented with dialyzed FBS. In certain embodiments, before the assay(e.g., 24-hours prior to assay), cells are switched to tyrosine-free,serum-free, exosome-free media so there is no detectable exosomecontamination from the medium alone.

In certain embodiments, the hypoxic oxygen environment is an environmentwith a concentration of oxygen of about 5%. In certain embodiments, thehypoxic oxygen environment is an environment with a concentration ofoxygen of about 10%. In certain embodiments, the hypoxic oxygenenvironment is an environment with a concentration of oxygen of about15%. In certain embodiments, the hypoxic oxygen environment is anenvironment with a concentration of oxygen from 2 to 10%. In certainembodiments, the hypoxic oxygen environment is an environment with aconcentration of oxygen from 5 to 15%.

In certain embodiments, the RPE cells are not placed in the hypoxicenvironment for a period of time prior to addition of the test compoundor drug, e.g., the test compound is added at the time the cells areplaced in the hypoxic oxygen environment.

In certain embodiments, the cells are incubated in a hypoxic O₂environment to induce exosome release for a time period between 30minutes to 4 hours. In certain embodiments, the cells are incubated in ahypoxic O₂ environment to induce exosome release for a time periodbetween 1 to 5 hours. In certain embodiments, the cells are incubated ina hypoxic O₂ environment to induce exosome release for a time periodbetween 4 to 10 hours. In certain embodiments, the cells are incubatedin a hypoxic O₂ environment to induce exosome release for a time periodbetween 5 to 15 hours. In certain embodiments, the cells are incubatedin a hypoxic O₂ environment to induce exosome release for a time periodbetween 12 to 24 hours. In certain embodiments, the cells are incubatedin a hypoxic 02 environment to induce exosome release for a time periodbetween 15 to 24 hours. In certain embodiments, the cells are incubatedin a hypoxic O₂ environment to induce exosome release for a time periodbetween 20 to 30 hours. The present invention is not limited to theaforementioned time periods.

In certain embodiments, the cells are incubated with the test compoundor drug for a time period from 10 to 30 minutes. In certain embodiments,the cells are incubated with the test compound or drug for a time periodfrom 20 to 30 minutes. In certain embodiments, the cells are incubatedwith the test compound or drug for a time period from 20 to 45 minutes.In certain embodiments, the cells are incubated with the test compoundor drug for a time period from 30 to 60 minutes. In certain embodiments,the cells are incubated with the test compound or drug for a time periodfrom 60 to 90 minutes. In certain embodiments, the cells are incubatedwith the test compound or drug for a time period from 1-3 hours. Incertain embodiments, the cells are incubated with the test compound ordrug for a time period greater than 3 hours. The present invention isnot limited to the aforementioned time periods.

In some embodiments, the present invention features a kit for screeningcompounds that are effective for treating age-related maculardegeneration (AMD). In some embodiments, the kit comprises: at least twocontainers for culturing and incubating retinal pigment epithelial (RPE)cells, RPE cells, tyrosine-free, serum-free, and exosome-free media, anda set of instructions for identifying a compound that is effective fortreating AMD. In further embodiments, the set of instructions comprises:culturing the RPE cells in each container with the media; artificiallyinducing exosome release from the RPE cells; adding the compound to theRPE cells in one of the containers; incubating the RPE cells in eachcontainer; and measuring a level of exosome release from the RPE cellsin each container. Without wishing to limit the present invention to anytheory or mechanism, if the level of exosome release from the RPE cellsin the container with the compound is at least 10% lower than the levelof exosome release from the RPE cells in the container without thecompound, then the compound is identified as being effective fortreating AMD. In some embodiments, the culture dish is a multi-wellplate. Non-limiting examples of multi-well plates include 96 well platesand 384 well plates. In other embodiments, the kit further comprisescomponents and instructions for isolating exosomes.

EXAMPLE

The following example describes the regulation of RPE-exosome releasewith L-DOPA. The present invention is not limited to the methods ormaterials described herein.

Exosomes serve as a critical inter-tissue communication and transportsystem, which may serve a vital role in RPE:retina interaction. Exosomesare small (40-100 nm diameter) extracellular vesicles produced withinthe multivesicular body (MVB), which is a multipurpose organelle of theendosomal system. The MVB fuses with the plasma membrane and releasesthe intraluminal vesicles as exosomes in a controlled manner.

Exosomes released from retinal pigment epithelial (RPE) cells can beisolated. For example, FIG. 2 shows a schematic view of exosomeisolation using differential ultracentrifugation. Cells and cell debrisare removed and exosomes are then purified. Note the present inventionis not limited to this particular method of exosome isolation. Incertain embodiments of the present invention, exosomes are not isolatedfor the measurement of levels of exosomes released from RPE cells.

Referring to FIG. 3A, RPE cells were incubated in either media (e.g.,DMEM) or media (e.g., DMEM) plus L-DOPA, showing a significant reductionin exosome release in the L-DOPA-treated RPE cells. FIG. 36 illustratesGPR143 exosome release and its inhibition by L-DOPA.

FIG. 4A and FIG. 4B show RPE exosome release studies in response to 30minute treatments with 1 μM dopamine (FIG. 4A), 1 nM Dopamine 2 receptor(D2R) agonist (bromocriptine, FIG. 46), and 1 nM Dopamine 2 receptor(D3R) antagonist (raclopride, FIG. 46). These studies illustrate thatstimulation of the D2R in the RPE reduces the release of exosomes.

FIG. 5 shows an exosome release study in response to hypoxia. RPE tissueisolates were cultured for 15 hours or 24 hours. Exosomes werequantified by unbiased protein staining of SDS-PAGE gels. The resultsshow that exosome release increases the longer RPE undergoes hypoxia.

Embodiments of the present invention can be freely combined with eachother if they are not mutually exclusive.

Although there has been shown and described the preferred embodiment ofthe present invention, it will be readily apparent to those skilled inthe art that modifications may be made thereto which do not exceed thescope of the appended claims. Therefore, the scope of the invention isonly to be limited by the following claims. In some embodiments, thefigures presented in this patent application are drawn to scale,including the angles, ratios of dimensions, etc. In some embodiments,the figures are representative only and the claims are not limited bythe dimensions of the figures. In some embodiments, descriptions of theinventions described herein using the phrase “comprising” includesembodiments that could be described as “consisting essentially of” or“consisting of”, and as such the written description requirement forclaiming one or more embodiments of the present invention using thephrase “consisting essentially of” or “consisting of” is met.

What is claimed is:
 1. A method of identifying a therapeutic compositioneffective for treating a condition caused by increased levels ofexosomes released from retinal pigment epithelial (RPE) cells, saidmethod comprising: a. artificially inducing exosome release fromcultured RPE cells; b. incubating the cultured RPE cells with atherapeutic composition; and c. measuring a level of exosome releasefrom the RPE cells; wherein if the level of exosome release from the RPEcells is lower than a predetermined threshold, then the therapeuticcomposition is identified as being effective for treating the condition.2. The method of claim 1, wherein the condition caused by increasedlevels of exosomes released from RPEs is age-related maculardegeneration (AMD).
 3. The method of claim 1, wherein the predeterminedthreshold is a level of exosome release measured from cultured RPE cellsin which exosome release was induced, and then incubated in mediawithout the therapeutic composition.
 4. The method of claim 1, whereinif the level of exosome release from RPE cells is at least about 10%lower than the predetermined threshold, the therapeutic composition isidentified as being effective for treatment.
 5. The method of claim 1,wherein media from the RPE cells that were incubated with thetherapeutic composition is collected for measuring the level of exosomerelease from the RPE cells.
 6. The method of claim 5 further comprisingisolating exosomes from the media collected.
 7. The method of claim 1,wherein measuring the level of exosome release from the RPE cells isperformed by measuring an absorbance at 260/280 (Ar) of media in whichthe RPE cells are cultured.
 8. The method of claim 1, wherein exosomerelease is induced by an artificial stressor.
 9. The method of claim 8,wherein the artificial stressor is a hypoxic environment or starvation.10. The method of claim 9, wherein the hypoxic environment is anenvironment with a concentration of oxygen between about 5-10%.
 11. Themethod of claim 1, wherein exosome release is induced for a first timeperiod ranging from about 12-24 hours.
 12. The method of claim 11,wherein the cultured RPE cells are incubated for a second time periodranging from about 15-60 minutes.
 13. The method of claim 1, wherein thetherapeutic composition is an activator of GPR143.
 14. A composition fortreating a condition caused by increased levels of exosomes releasedfrom retinal pigment epithelial (RPE) cells, wherein said composition isidentified for treating the condition by: a. inducing exosome releasefrom cultured RPE cells; b. incubating the cultured RPE cells with atherapeutic composition; and c. measuring a level of exosome releasefrom the RPE cells, wherein if the level of exosome release from the RPEcells is lower than a predetermined threshold, then the therapeuticcomposition is identified as being effective for treating the condition.15. A method of preventing progression of dry age-related maculardegeneration (AMD) to wet AMD in a subject in need thereof, said methodcomprising: administering to the subject a therapeutically effectiveamount of a composition identified in claim
 14. 16. The method of claim15, wherein the therapeutic composition blocks or reduces exosomerelease in retinal pigment epithelial (RPE) cells.
 17. A method oftreating age-related macular degeneration (AMD) in a subject in needthereof, said method comprising: administering to the subject atherapeutically effective amount of a composition identified in claim15.
 18. A kit for screening compounds that are effective for treatingage-related macular degeneration (AMD), said kit comprising: a. at leasttwo containers for culturing and incubating retinal pigment epithelial(RPE) cells; b. RPE cells; c. tyrosine-free, serum-free, andexosome-free media; and d. a set of instructions for identifying acompound that is effective for treating AMD, said instructionscomprising: i. culturing the RPE cells in each container with the media;ii. artificially inducing exosome release from the RPE cells; iii.adding the compound to the RPE cells in one of the containers; iv.incubating the RPE cells in each container; and v. measuring a level ofexosome release from the RPE cells in each; wherein if the level ofexosome release from the RPE cells in the container with the compound isat least 10% lower than the level of exosome release from the RPE cellsin the container without the compound, then the compound is identifiedas being effective for treating AMD.
 19. The kit of claim 18 furthercomprising components and instructions for isolating exosomes.